West Nile Virus Infection: Novel Genetic Screens to Identify Important Host Genes

描述（由申请人提供）：迫切需要更好地了解西尼罗河病毒（WNV）的地方性传播周期，特别是了解宿主基因在确定不同物种和品系蚊子对WNV感染的易感性为何不同以及这种变化如何影响疾病传播方面所起的作用。本研究的长期目标是了解虫媒病毒与其宿主之间的分子相互作用如何决定感染易感性。目前系统鉴定蚊子宿主基因的一个主要障碍是在蚊子中进行功能性遗传筛选是不切实际的，这些基因在西尼罗河病毒感染中是重要的。本项目的目的是评估使用无偏见的，功能性的遗传筛选在果蝇作为一种替代的实验方法，以确定在宿主基因突变的重要西尼罗河病毒感染的效用。这一目标的前提是假设使用D。黑腹果蝇作为西尼罗河病毒感染的替代宿主将提供一个遗传上易于处理的模型系统，以加速发现对感染重要的宿主基因。这一假设是基于初步研究表明，D。黑腹果蝇可被西尼罗河病毒感染。D.黑腹果蝇鉴定对WNV感染重要的宿主基因将为鉴定调节病毒的重要蚊子载体中的感染易感性的蚊子基因提供基于经验的起点。提出了一个具体的目标：鉴定D.影响西尼罗河病毒感染苍蝇的能力。这一目标是基于工作假设，即D。涉及WNV感染的黑腹菌基因将导致病毒滴度容易检测到改变的感染。提出了两种遗传筛选，其中EMS诱导的隐性突变或P元件诱导的错误表达突变将筛选其对WNV感染的影响，通过将病毒注射到含有诱变染色体或全基因组P元件插入的果蝇中，诱导邻近基因的错误表达，并通过RT-PCR定量WNV基因组RNA的产生。这些研究是有意义的，因为它们可能提供一个原理证明，即在D。melanogaster基因可用于克服由WNV的天然蚊子宿主呈现的技术限制，以鉴定对WNV感染重要的宿主基因。 公共卫生相关性：有一个遗传学上易于处理的模型系统，调查主机：虫媒病毒的相互作用将大大有助于确定宿主基因的重要西尼罗河病毒感染，并将提供工具，阐明这些基因如何影响地方病传播周期，并最终，疾病传播。在确定这些宿主基因之前，对确定感染易感性重要的宿主：西尼罗河病毒相互作用的充分理解仍然有限，阻碍了制定有效战略以减少西尼罗河病毒疾病的出现和传播所造成的健康负担的努力。

DESCRIPTION (provided by applicant): There is a critical need to better understand the enzootic transmission cycles of West Nile virus (WNV), particularly as relates to understanding the role that host genes play in determining why different species and strains of mosquito vary in their susceptibility to WNV infection and how such variation impacts disease transmission. The long term goal of this study is to understand how the molecular interactions that occur between arboviruses and their hosts determine infection susceptibility. A major current impediment to the systematic identification of mosquito host genes that are important in WNV infection is the impracticality of doing functional genetic screens in mosquitoes. The objective of this project is to evaluate the utility of using unbiased, functional genetic screens in Drosophila melanogaster as an alternative experimental approach to identifying mutations in host genes important for WNV infection. This objective is predicated on the hypothesis that using D. melanogaster as a surrogate host for WNV infection will provide a genetically tractable model system to expedite the discovery of host genes important for infection. This hypothesis is based on preliminary studies demonstrating that D. melanogaster can be infected by WNV. Successful utilization of D. melanogaster to identify host genes important for WNV infection would provide an empirically based starting point for identifying mosquito genes that modulate infection susceptibility in important mosquito vectors of the virus. One specific aim is proposed: identify mutations in D. melanogaster that affect the ability of WNV to infect flies. This aim is based on the working hypothesis that mutations in D. melanogaster genes involved in WNV infection will lead to infections that have readily detectable alterations in virus titer. Two genetic screens are proposed in which EMS-induced recessive mutations or P element-induced misexpression mutations will be screened for their effects on WNV infection by injecting virus into flies containing either mutagenized chromosomes or genome-wide P-element insertions that induce the misexpression of neighboring genes and quantifying the production of WNV genomic RNA by RT-PCR. The proposed studies are significant, because they may provide a proof-of- principle demonstration that the powerful genetic methodologies available in D. melanogaster can be used to overcome technical limitations presented by natural mosquito hosts of WNV in order to identify host genes important for WNV infection. Public Health Relevance: Having a genetically tractable model system to investigate host:arbovirus interactions would greatly facilitate the identification of host genes important for WNV infection and would provide the tools for the elucidation of how those genes impact enzootic transmission cycles and, ultimately, disease transmission. Until such host genes have been identified, a full understanding of host:WNV interactions that are important for determining infection susceptibility will remain limited, impeding efforts to develop effective strategies for reducing the health burden caused by the emergence and spread of WNV disease.